Cell Culture
Figure 1
Spheroids show increased sensitivity to DILI compound treatment versus 2D cultures
l 3D cell cultures provide more architecturally rel- evant barriers for compounds to traverse than pre- sent in 2D cell cultures. Tissue absorption is a major factor in determining efficacy and many compounds have similar access to all cells in flat 2D cultures, resulting in overestimation of efficacy. l Mixed cell type 3D culture systems allow better modelling of cell-cell interactions than is possible with 2D cell culture systems and can even maintain neuronal inputs to the organ or organoid. l 3D cell cultures can be long-lived, in some cases for months. This temporal aspect of cell culture is important when modelling diseases that are slow to develop, such as neurodegenerative diseases. The relatively long life of the culture provides a system to identify pathological defects emerging in the tissue over time, as well as monitor the prolonged expo- sure of cells to pharmacologically active com- pounds.
Drug-Induced Liver Injury (DILI) assessment using 3D hepatocyte cultures A comparison of 2D and 3D primary human hep- atocytes for assessment of drug compounds to cause liver injury demonstrates the advantages of 3D approaches. Drug-induced liver injury (DILI) is a leading cause of drug attrition and clinical failure and can manifest as mitochondrial dysfunction, apoptosis, necrosis, hepatocyte hypertrophy and hyperplasia, fibrosis and cholestasis. The impact of DILI on drug development is clear – among the
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drugs withdrawn due to toxicity during the period of 1990-2010, 26% were attributed to DILI. While 2D cell culture systems play a pivotal role
in research, classical systems do not reflect the com- plexity of liver tissue. Although hepatocytes are often the in vivo targets of DILI, primary human hepatocytes are not suitable for hepatotoxicity tests under conventional 2D monolayer culture condi- tions, due to the rapid loss of their hepatic pheno- types, functions and cell viability. In contrast, 3D cell culture systems sustain cell viability, maintain in vivo phenotypes as well as genomic and proteomic expression profiles. Compared with other 3D sys- tems, spheroid hepatocyte cultures require fewer cells, are technically easier to establish, and are adaptable to high throughput screening. There are several well-established protocols,
tools and reagents to support primary 3D liver spheroids and toxicity assays. Single spheroids can be generated using seeding densities with less than 5,000 cells per well on 96-well spheroid plates. Morphology and sizes can be routinely monitored and bioluminescent ATP assays for viability can be performed directly in the spheroid microplate. The primary human hepatocytes maintain Cytochrome P450 (CYP) drug metabolic activity in long-term cultures of up to four weeks. In contrast, 2D human hepatocyte cultures are short-lived, typical- ly lasting only one week. As shown in Figure 1, primary human hepato-
cyte spheroids are more sensitive than 2D cultures for detecting liver toxicity. Amiodarone, an
Drug Discovery World Winter 2018/19
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